Lipid A is a mixture of at least six species of molecules whose struc-tures differ both in the phosphorylation of sugar backbone and in fatty acylation.. All lipid A species carry four am
Trang 1Characterization of Mesorhizobium huakuii lipid A containing both
Adam Choma1and Pawel Sowinski2
1
Department of General Microbiology, Maria Curie-Sklodowska University, Lublin, Poland;2Intercollegiate NMR Laboratory, Department of Chemistry, Technical University of Gdansk, Poland
The chemical structure of the free lipid A isolated from
Mesorhizobium huakuiiIFO 15243Twas elucidated Lipid A
is a mixture of at least six species of molecules whose
struc-tures differ both in the phosphorylation of sugar backbone
and in fatty acylation The backbone consists of a b (1¢fi 6)
linked 2,3-diamino-2,3-dideoxyglucose (DAG) disaccharide
that is partly substituted by phosphate at position 4¢ The
aglycon of the DAG-disaccharide has been identified as
a-D-galacturonic acid All lipid A species carry four
amide-linked 3-hydroxyl fatty residues Two of them have short
hydrocarbon chains (i.e 3-OH-i-13:0) while the other two
have longer ones (i.e 3-OH-20:0) Distribution of 3-hydroxyl
fatty acids between the reducing and nonreducing DAG is symmetrical The nonpolar as well as (x-1) hydroxyl long chain fatty acids are components of acyloxyacyl moieties Two acyloxyacyl residues occur exclusively in the non-reducing moiety of the sugar backbone but their distribution has not been established yet The distal DAG amide-bound fatty acid hydroxyls are not stoichiometrically substituted
by ester-linked acyl components
Keywords: Mesorhizobium huakuii; lipid A; 2,3-diamino-2,3-dideoxy-D-glucose; MALDI-TOF; 2D-NMR
Lipopolysaccharides (LPS) are characteristic components of
the outer leaflet of the outer membranes of Gram-negative
bacteria Those glycoconjugates have a common general
architecture They contain three distinct regions: lipid A, a
nonrepeating oligosaccharide core and an O-polysaccharide
composed of a varying number of repeating units The
O-polysaccharide chain is the major target of animal
immune responses, thus it is also referred to as the
O-antigen The core oligosaccharide is a spacer between
the O-chain and lipid A and is linked to the latter by an acid
labile ketosidic bond Lipids A in many Gram-negative
bacteria (especially in animal pathogens) have a conserved
structure In the majority of cases, their backbones are
composed of a b-1,6-D-glucosamine disaccharide with two
phosphate residues attached at positions 1 and 4¢ Up to
four fatty acids are bound by ester or amide linkages
to the backbone glucosamines Lipid A is responsible for the
endotoxic properties of lipopolysaccharide The structure of
lipid A seems to be essential in maintaining outer membrane
integrity and flexibility and is crucial for bacterial cell
viability [1–3]
Lipopolysaccharide is important in the process of
sym-biotic interaction between Rhizobium and the host plant
[4,5] Environmental conditions (in planta and ex planta) as
well as plant-derived molecular signals induce entire LPS modifications in Rhizobium [6]
The structures of Rhizobium lipid A indicate great variation in the glycosyl component of its backbone as well
as the acylation pattern The lipid A backbone of Sinorhizo-biumis similar to that from enteric bacteria [7,8] Lipids A from Rhizobium etli and biovars of Rhizobium legumino-sarumhave identical and unusual structures R etli lipids A are devoid of phosphate groups [9–11] and a galacturonic acid residue replaces the 4¢-linked phosphate in the lipid A backbone The distal part (distant from the reducing end of the backbone) of lipid A is almost the same for all lipid A species isolated The proximal glucosamine is partly oxi-dized to 2-aminogluconate [12,13] A specific deacylase removes the ester-linked fatty acids from the C-3 position
of the lipid A precursor, thus this hydroxyl is only partially substituted by an acyl residue in the matured lipid A [14] The symbiont of Sesbasnia, Rhizobium sp Sin-1 [15], has lipid A composed of b-D-glucosamine attached to 2-aminogluconate by (1fi 6) glycoside linkage When compared with R etli this lipid A lacks galacturonic acid
at position 4¢ [16]
In contrast to the above mentioned lipid A structures, the mesorhizobial and bradyrhizobial lipids A have not been fully chemically characterized to date Bradyrhizobium lipid A backbones are composed exclusively of 2,3-di-amino-2,3-dideoxyglucose with mannose as a subsituent in some of them [4,5,17,18] No data about Allorhizobium (renamed Rhizobium undicola [19]) and scant information about Azorhizobium [20,21] lipopolysaccharides and lipids A are available Mesorhizobium loti lipids A contain DAG and phosphate residues [22,23] and M huakuii also posses-ses DAG-type lipid A [24] Mesorhizobium lipids A are known to carry a number of b-hydroxyl fatty acids accompanied by small amounts of 4-oxo fatty acids
Correspondence to A Choma, Department of General Microbiology,
Maria Curie-Sklodowska University, 19 Akademicka St.,
20–033 Lublin, Poland.
Fax: + 48 81 5375959, Tel.: + 48 81 5375981,
E-mail: achoma@biotop.umcs.lublin.pl
Abbreviations: DAG, 2,3-diamino-2,3-dideoxyglucose; LPS,
lipopolysaccharides.
(Received 12 September 2003, revised 6 February 2004,
accepted 16 February 2004)
Trang 2Numerous ester-linked nonpolar and (x-1) hydroxyl long
chain fatty residues were found in those preparations [25,26]
In this report, we describe the structural investigation of a
unique lipid A isolated from Mesorhizobium huakuii We
show that DAG-type lipid A backbone is double decorated:
(a) nonstoichiometrically, with phosphate at position 4¢ of
the distal DAG, and (b ) with a-linked galacturonic acid
at position 1 of the proximal unit Phosphorylated and
nonphosphorylated lipid A preparations are a mixture of
three subfractions differing in acylation patterns
Experimental procedures
Bacterial strain, growth, and isolation of
lipopolysaccharide and lipid A
Mesorhizobium huakuii IFO15243T strain was obtained
from the Institute for Fermentation, Osaka, Japan Bacteria
were grown at 28C in liquid mannitol/yeast extract
medium 79CA [27] and were aerated by vigorous shaking
Cells were centrifuged at 10 000 g, washed twice with saline
and once with distilled water The wet bacterial paste was
extracted by the modified hot phenol/water procedure [28]
The water layer was dialysed firstly against tap water, then
against distilled water The crude LPS was purified by
repeated ultracentrifugation at 105 000 g for 4 h The LPS
solution (5 mgÆmL)1) in aqueous 1% (v/v) acetic acid was
kept at 100C for 3 h The lipid A precipitate was collected
by centrifugation, washed twice with hot distilled water and
lyophilized
Purification and separation of lipid A species
Crude lipid A was purified and separated into subfractions
according to a modified procedure described by Que and
coworkers [9] Briefly, lyophilized lipid A ( 30 mg) was
dissolved in 20 mL of CHCl3/methanol/H2O (2 : 3 : 1;
v/v/v) and loaded onto a DEAE column (1 cm· 7 cm,
Whatman DE23) The column was washed with 30 mL of
the same solvent and that eluate was collected as a single
fraction Next, the lipid material was eluted by a two step
gradient of ammonium acetate: first with 30 mL of CHCl3/
methanol/250 mMNH4Ac (2 : 3 : 1; v/v/v), and then with
30 mL of CHCl3/CH3OH/500 mM NH4Ac (2 : 3 : 1;
v/v/v) The presence of organic substances in the eluate
was monitored by spotting 10 lL of each fraction on a silica
plate and visualized by spraying the plate with 10% (v/v)
sulfuric acid in methanol followed by charring Separated
fractions were converted to two-phase Bligh–Dyer system
by adding the appropriate amount of water and chloroform
Water layers were discarded and organic layers were
supplemented with fresh portions of the upper phase of
a freshly prepared two-phase Bligh–Dyer mixture The
washed organic layers were separated by centrifugation and
dried Preparations were stored at )20 C in CHCl3/
methanol (1 : 1; v/v)
Glycosyl composition analysis
Lipid A samples were analysed for fatty acids and
amino-sugars as described previously [24] Neutral and acidic
sugars were determined by gas-liquid chromatography and
mass spectrometry For this analysis, lipid A samples were methanolysed (1M HCl, 80C, 18 h), N-acetylated and trimethylsililated [29] The content of phosphorus in lipid A was determined according to Lowry [30]
Chemical modification of lipid A Subfractions of lipid A (about 2 mg) were dephosphory-lated in 48% (v/v) aqueous HF at 4C for 48 h [31] HF was removed by evaporation in the stream of nitrogen with cooling in an ice bath De-O-acylation of lipid A subfractions was performed according to modified procedure of Haishima and coworkers [31] Preparations were treated with anhy-drous hydrazine at 37C for 2 h The reaction mixtures, after cooling, were poured into cold acetone The resulting lipid A precipitates were collected, washed twice with acetone and then gently dried in the stream of nitrogen
Gas chromatography-mass spectrometry GC-MS was carried out on a Hewlett-Packard gas chromatograph (model HP5890A) equipped with a capil-lary column (HP-5MS, 30 m· 0.25 mm) and connected to
a mass selective detector (MSD model HP 5971) Helium was the carrier gas The temperature program for fatty acid methyl esters and for alditol acetates analysis was as follows: initially 150C for 5 min, then raised to 310 C at a ramp rate of 3C min)1, final time 20 min The temperature program for trimethylsililo derivatives of methyl glycosides was, accordingly: initially 80C for 2 min, then raised to
310C at a ramp rate of 4 C min)1, final time 5 min Mass spectrometry
Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was performed on a Voyager-Elite (PE Biosystems) instrument using delayed extraction, in both positive and negative ion modes The samples were desorbed with a nitrogen laser and extraction voltage of 20 kV Lipid A samples were dissolved in CHCl3/
CH3OH (2 : 1; v/v) The analysed compounds (0.5 lL) were mixed with 50% (v/v) 2,5-dihydrobenzoic acid in acetonitril as matrix Each spectrum was the average of about 256 laser shots
Liquid matrix-assisted secondary ion mass spectrometry (LSIMS) was performed using AMD 604 (AMD Intectra GmbH) mass spectrometer operated in the negative ion mode with primary ion beam of Cs+ Samples were mixed with a matrix of meta-nitrobenzyl alcohol (m-NBA) Lipid A was analysed by ESI-MS using Finnigan Mat TSQ 700 mass spectrometer operated in the negative ion mode The samples were dissolved in a CHCl3/CH3OH (2 : 1; v/v) mixture supplemented with 0.1% (v/v) concen-trated ammonia and introduced into electrospray source
at a flow rate of 5 lLÆmin)1 NMR spectroscopy
1H-NMR experiments were performed in CDCl3 /dimethyl-sulfoxide-d6(2 : 1; v/v) mixture 2D (DQF COSY, TOCSY, NOESY)1H-NMR and 1H/13C as well as1H/31P-HSQC experiments were carried out on Varian Unity plus 500
Trang 3instrument at 48C using standardVARIANsoftware 1D
31P-NMR spectra were registered on a Bruker 300
spectro-meter, operating at 121.58 MHz at 40C For this analysis,
the lipid A was dissolved in D2O containing 2%
deoxycho-late and 5 mMNa2EDTA The pH of lipid A solutions was
adjusted with NaOH to 7.3 and 10.6, respectively
Phospho-rous chemical shifts were measured relative to an external
standard of 85% (v/v) phosphoric acid at 0.00 p.p.m
Results
Chemical analyses
The compositional analysis of crude lipid A preparation
obtained from M huakuii IFO 15243T LPS revealed the
presence not only of 2,3-diamino-2,3-dideoxyglucose
(DAG) and a complex set of fatty acids (both ester and
amide bound), as described previously [24], but also
galacturonic acid and phosphate residues The presence of
GalA was unequivocally confirmed by GC-MS analysis of
trimethylsilil ethers of methyl glycosides liberated from
lipid A by methanolysis (Fig 1) The31P-NMR spectrum
of the crude lipid A revealed a prominent signal with
chemical shift of 1.71 p.p.m observed at neutral pH This
signal was shifted to 4.71 p.p.m when the pH of the lipid A
suspension was raised to 10.6 (Fig 2) These properties are
indicative of phosphomonoesters other than
glycosyl-1-phosphate The location of the phosphate was directly
determined by two-dimensional heteronuclear magnetic
resonance (see below) On the basis of chemical shift value
and lack of the cross peak with protons from the lipid A
backbone on the31P/1H-HSQC spectrum, the weak signal
at 1.88 p.p.m was attributed to inorganic phosphate
impurities of the lipid A preparation The results of
quantitative measurements of phosphorus and DAG
con-tent showed that no more than half of the lipid A molecules
bear phosphate residues
Fatty acids found in the IFO 15243Tlipid A (Fig 1) can
be divided into two groups The first one, easily liberated by
mild alkali or acid solvolysis, contains all saturated and
unsaturated nonpolar as well as (x-1) hydroxyl, and (x-1)
oxo long chain fatty acids These are ester-linked to the lipid A The second group of fatty acids needs strong liberation conditions [32] This group comprises all 3-hydroxyl and 4-oxo fatty acids, which are connected directly to the lipid A backbone via amino groups [24] The molar proportions among fatty acids isolated from lipid A were almost the same as described earlier for the total LPS [24] The main amide-bound fatty acids identified were as follows: 12:0, i-13:0, 20:0 and 3-OH-21:0 Among them, 3-OH-i-13:0 and 3-OH-20:0 clearly predominated The types of ester-bound fatty acids were also numerous, but only four of them, namely i-17:0, 20:0, 22:1 and particularly 27-OH-28:0 fatty acid, predomi-nated (Fig 1, [24]) The calculated proportion between
Fig 1 GC-MS profile of trimethylsilyl ether derivatives of N-acetylated methyl glycosides and fatty acid methyl esters obtained by methanolysis of dephosphorylated lipid Afrom Mesorhizobium huakuii IFO 15243T Peaks were identified by their mass spectra and by comparison of retention times with standards GalA, galacturonic acid; DAG, 2,3-diamino-2,3-dideoxyglucose; p.e impurity-ester of phtalic acid; *, unidentified compound For more details about the fatty acid composition of IFO 15243Tsee [24].
Fig 2.31P-NMR spectra of the crude lipid Afrom Mesorhizobium huakuii IFO 15243T The signal at 1.71 p.p.m (A) recorded at pH 7.3, shifted to 4.71 p.p.m (B) at pH 10.60, and represents ester-bound monophosphate residue.
Trang 4amide- and ester-linked fatty acids was approximately 2 : 1.
Therefore, one can expect that DAG type lipid A could
contain not more than six acyl residues
The complex mixture of the lipid A preparation was
separated into two fractions, based on DEAE gravity
column chromatography The first fraction (designated
lipid A–P), which was eluted with solvent containing
250 mMammonium acetate, was devoid of phosphate, as
shown by 31P-NMR The phosphate was detected in
the second fraction (named lipid A+P), successively eluted
with a solvent mixture containing 500 mMNH4Ac
MALDI-TOF analysis of lipid A preparations
Both subfractions of lipid A were investigated by mass
spectrometry Ions representative of each species of lipid A,
recorded on the negative and positive ion MALDI-TOF
and negative ion ES-MS spectra, their corresponding
composition and the theoretically calculated masses are
listed in Table 1
Lipid A+Pis a complex mixture of individual molecules
The two major species (Z and Y) could be easily
distinguished from mononegatively charged
pseudomole-cular ions on MALDI-TOF spectrum (Fig 3A) The third
(X) cluster of ions, which were less intensive, was visible
between m/z at 1600 and 1700 All those ions correspond to
lipid A that posseses a backbone with a monophosphate
residue accompanied by six, five and four acyl moieties,
respectively Dephosphorylation procedure, to which
lipid A+P was submitted, led to a downshift of each
molecular ion by 80 mass units The spectrum of the
dephosphorylated lipid A+P is almost identical to that
obtained for the lipid A–Ppreparation (Fig 4) Moreover,
the MALDI-TOF spectrum of lipid A–Ptreated with 48%
HF did not change significantly when compared with the
unprocessed preparation (data not shown)
Species Z of lipid A+P(Fig 3A) contained ions within the
range from 2287 to 2478 mass units Those ions correspond
to lipid A molecules composed of two DAG, one of which
is phosphorylated, one GalA, four 3-hydroxyl fatty acids,
one (x-1) hydroxyl long chain fatty acid and one a nonpolar
fatty acyl residue The most intense ion in this cluster (m/z
at 2357) could be attributed to the molecules of lipid A
containing two 3-OH-i-13:0 and two 3-OH-20:0 acids, as
well as two ester-bound acids (e.g 20:0 and 27-OH-28:0)
This is merely one possible explanation due to the fact that
numerous combinations of fatty acids different to those
found in lipid A exist However, taking into consideration
the quantities of lipid A fatty acids this proposition seems
to be the most probable The amide-bound fatty acids
isolated from M huakuii IFO15243T lipid A and from
other mesorhizobia can be separated into two clusters
[24–26] The first contains short chain fatty acids, mainly
3-OH-12:0 and 3-OH-i-13:0, whereas the second is
repre-sented by 3-OH-20:0 and other fatty acids similar in length
For correct calculation of the pseudomolecular ion masses
found on the MALDI-TOF spectra it is necessary to take
into account the masses of two 3-OH short chain fatty
acyls (e.g 3-OH-i-13:0) and two longer 3-OH fatty acyl
residues (e.g 3-OH-20:0)
The ions from species Y are usually 295 mass units
lighter than the respective ions from species Z That
corresponds to a loss of eicosanoyl residue from hexaacyl lipid A Therefore, the Y species comprise ions represent-ing lipid A molecules carryrepresent-ing five acyl residues (four 3-OH fatty acids and one (x-1) hydroxyl long chain fatty acid) The ion at m/z 1640 and those close to m/z 1640, designated as species X, correspond to tetraacyl lipid A molecules with all acyl residues directly linked to the sugar backbone by amide bonds De-O-acylation of lipid A fractions led to decay of species Z and Y and resulted in increase of signals corresponding to ions of species X (data not shown) The total decrease of mass due to de-O-acylation of phosphorylated as well as of nonphospho-rylated lipid A was the same and equalled 717 Da (loss of both 294 and 423 mass units)
The positive ion MALDI-TOF mass spectra of the lipid A+P(Fig 3B) showed two additional species gener-ated after laser-induced cleavage of glycosidic linkages between 2,3-diamino-2,3-dideoxyglucoses within the lipid
A backbone The first species [B1+(Z)] of oxonium ions originated from hexaacyl phosphorylated lipid A (pro-minent ions at m/z 1481 and 1508) The second species [B1+(Y)] consist of ions with masses close to that at m/z
1187 Those ions are made up of DAG, two 3-hydroxyl fatty acyl moieties and (x-1) hydroxyl long chain fatty acid Those B1+fragment ions support the conclusion that the 27-hydroxyoctacosanoic acid and eicosanoic acid, when present, are located on the distal diaminoglucosyl residue of the lipid A Moreover, the sugar component of
B1+ lacks hydroxyl groups suitable for attachment of these fatty acids by ester bonds The appropriate hydroxyls are located at positions 3 of amide linked acyl of the distal DAG Therefore, both 27-OH-28:0 and 20:0 fatty acids are components of acyloxyacyl residues The predicted ions for the third type of oxonium ions composed of DAG and two amide acyl residues have not been registered, due to the fact that the spectra were usually recorded from m/z 1000–3000 The correct calculation of masses for B1+type ions requires taking into account the appropriate amide-linked fatty acids That group of acyl residues consists of fatty acid pairs The first acid in each pair is shorter (e.g i-13:0) while the second one is longer (e.g 3-OH-20:0) Analysis of lipid A by means of LSI mass spectro-metry revealed negative ions m/z at 862.7, 876.8 and 890.7 (data not shown) The most intensive ion (m/z at 876.8) corresponds to a lipid A fragment composed of DAG, GalA, 3-OH-i-13:0 and 3-OH-20:0 A similar ion was observed for P gingivalis and F meningosepticum lipids A
on negative ion FAB-MS/MS spectra [33,34] In conclu-sion, these data point to the symmetrical localization of amide-bound acyl residues in M huakuii lipid A The 2,3-diacylamido-2,3-dideoxyglucose, obtained by mild solvolysis [35] followed by mild hydrolysis of the dephos-phorylated lipid A, was reduced with NaBD4, than subjected to Smith oxidation, again reduced with NaBH4 and after acetylation, the four-carbon fragments of DAG carrying amide-bound fatty acids were analysed by means
of GC-MS Preliminary data from those experiments indicate that N-2 position in distal and proximal DAG is occupied mainly by 3-hydroxyleicosanoic acids The shor-ter acids were found to be bound at N-3 position of the amino sugar ring The fatty acid distribution will be verified during further studies
Trang 5T ;t
ES-MS (ion
-fatty acids
fatty acids
P-DAG, 1 ·
P-DAG, 1 ·
DAG 2 ·
DAG 2 ·
Trang 6The B1+ions from lipid A+P(e.g m/z at 1187 and 1508,
Fig 3B) differed by 80 mass units from those originating
from lipid A–P(e.g m/z at 1108 and 1428, Fig 4)
Comparing Figs 3B and 4, it is easy to notice that
the phosphate deprived lipid A appears to have a higher
number of connected fatty acids On the spectrum,
shown in Fig 4, the signals for hexaacyl lipid A are
considerably more intensive than others Pentaacyl
lipid A molecules dominate in the case of the phos-phorylated lipid A preparation Possibly, a weak acid hydrolysis (the procedure used for lipid A liberation) causes a partial de-O-acylation of the native lipid A molecules
In contrast to R etli, R leguminosarum and S melilotii [8–10], we did not find lipid A molecules containing 3-hydroxylbutyrate or 3-metoxylbutyrate
Fig 3 Negative (A) and positive (B) ion MALDI-TOF mass spectra of the phosphorylated subfraction of lipid A from M huakuii IFO 15243T Lipid A yields three ion clusters (Z, Y, X) They differ by the degree of acylation Species X contains four amide-bound fatty acids Species Y is pentaacyl lipid A (with 27-OH-28:0 fatty acid residue) Species Z is hexaacyl lipid A The proposed formulas and masses of pseudomolecular ions ([M ) H] – and [M + Na] + ) are summarized in Table 1 The individual ions in the clusters differ by 14 units (acyl chain length differences) Positive ion spectrum contains two B+1 type ion clusters derived from cleavage of the glycosidic linkage in lipid A Unidentified ions are marked with asterisks (*).
Trang 7NMR spectroscopy of lipid A preparations
De-O-acylated lipids A+Pand lipid A–Pwere dissolved in a
mixture of dimethylsulfoxide (DMSO-d6) and chloroform
(CDCl3) for NMR experiments Figure 5 shows the
one-dimentional proton spectrum of de-O-acylated lipids A+P
1H and13C chemical shift assignments were based on 2D
homonuclear experiments: DQF-COSY (Fig 6), TOCSY
(Fig 7) and 1H/13C heteronuclear single quantum
coher-ence (HSQC) experiments The values of carbon and proton
chemical shifts are summarized in Table 2
Three signals were identified in the anomeric region of
13C-NMR chemical shifts for both lipid A fractions These
data suggested that the lipid A backbone contains three
sugar residues Four signals were found between 50 and
55 p.p.m for each preparation They were assigned to the
C-2 and C-3 carbon atoms linked directly with the amino
groups The remaining sugar ring carbon signals were
observed in the region from 60 to 78 p.p.m TOCSY and
DQF-COSY spectra revealed three glycosyl ring systems
The anomeric proton (HA-1) at 4.98 p.p.m was assigned to a-linked galacturonic residue Its spin system (A) consists of five protons for which all the cross peaks have been traced and marked on Fig 6 and resulting chemical shifts listed in Table 2 Analysis of the sugar proton system B (Fig 7) was initiated at the anomeric proton (HB-1, dH¼ 4.87 p.p.m.,
J1,2¼ 2.8Hz) That proton showed an evident correlation
to HB-2 (dH¼ 3.84 p.p.m.), which showed a strong corre-lation to HB-3 (dH¼ 4.08 p.p.m.) Furthermore, HB-3 showed a coupling with HB-4 (dH¼ 3.48 p.p.m.) The remaining glycosyl proton cross-peaks were observed at following chemical shifts: 3.48 p.p.m./3.97 p.p.m (HB-4/
HB-5), 3.97 p.p.m./3.60 p.p.m (HB-5/HB-6a), 3.60 p.p.m./ 3.89 p.p.m (HB-6a/HB-6b) The proton chemical shifts for both sugar ring systems (A and B) were similar to those published for A pyrophilus lipid A [36] Chemical shifts of the distal aminosugar (sugar ring system C) in lipid A+P were in good agreement with those from A pyrophilus lipid A distal DAG, however, two shift exceptions (for HC-4 and HC-3) were observed The HC-4 signal appeared at
Fig 5 Proton NMR spectrum of de-O-acylated lipids A +P fraction The sample was dissolved in DMSO-d 6 /CDCl 3 (1 : 2, v/v) The spectrum was recorded at 500 MHz, at 48 C Some signals from sugar backbone are indicated The letters refer to the carbohydrate spin systems as was described
in the text and shown in Table 2 The numerals next to the letters indicate the protons in the respective residues Signal positions from olefinic protons, terminal methyl protons, bulk methylene protons and protons from a, b and c positions of 3-hydroxy fatty acids are marked with; -CH ¼ CH-, -CH , and -CH -, a, b and c, respectively CHCl , DMSO and H O represent signals from solvents and absorbed water.
Fig 4 Positive ion MALDI-TOF mass spectrum of unphosphorylated subfraction of lipid A from M huakuii IFO 15243 T This lipid A subfraction yields the three ion clusters X1, Y1 and Z1 They differ in the degree of acylation pattern and contain four, five and six acyl residues, respectively The spectrum contains two B +
1 type ion clusters derived by cleavage of the glycosidic linkage in lipid A.
Trang 8dH¼ 4.01 p.p.m., which was about 0.3 p.p.m downfield
from the A pyrophilus lipid A equivalent signal and about
0.5 p.p.m downfield from the H-4 signal characteristic of
DAG with unsubstituted hydroxyl group at C-4 carbon
atom (dHfor HB-4, Table 2) The downfield shift of HC-4
was caused by the presence of ester-bound phosphate
residue Analysis of carbon chemical shifts led to the same
conclusions, since C -4 (d ¼ 71.9 p.p.m) appeared
down-field compared to the proximal CB-4 unsubstituted by phosphate (dB¼ 67.5 p.p.m) The location of phosphate substituent on CC-4 was established upon HC-4/31P (4.01 p.p.m./1.35 p.p.m) correlation observed in 1H/31P HSQC spectrum
The sequence of the monosaccharides was established
by NOESY experiment (Fig 8) A strong interresidue NOE signal was observed between H -1 of GalA and
Fig 6 Apartial DQF-COSY spectrum of de-O-acylated phosphorylated subfraction of lipid A The spectrum was recorded at 500 MHz, at 48 C The letters refer to the carbohydrate spin systems as was described in the text and shown in Table 2 The numerals next to the letters indicate the protons in the respective residues.
Fig 7 Apartial TOCSY spectrum of de-O-acylated phosphorylated subfraction of lipid A The spectrum was recorded at 500 MHz, at 48 C The letters refer to the carbohydrate spin systems as was described in the text and shown in Table 2 The numerals next to the letters indicate the protons
in the respective residues.
Trang 9Table 2 1H- and 13C-NMR chemical shifts and coupling constants of sugar backbones of lipid Afractions DAG-I proximal 2,3-diamino-2,3-dideoxyglucose moiety in the lipid A from M huakuii IFO 15243 T , DAG-II distal 2,3-diamino-2,3-dideoxyglucose moiety in the lipid A from
M huakuii IFO 15243 T ; lipid A +P , phosphorylated fraction of lipid A; lipid A –P , unphosphorylated lipid A; nd, not determined; J, coupling constant Spectra were recorded at 500 MHz (1H) and 125.7 MHz (13C) in DMSO-d 6 /CDCl 3 (2 : 1, v/v).
Residue
(spin system)
1 H d (J,[Hz]) 13 C d 1 H d (J,[Hz]) 13 C d 1 H d (J,[Hz]) 13 C d Lipid A+P
H-1 4.39
(8.2)
C-1 103.1 H-1 4.87
(2.8)
C-1 92.8 H-1 4.98
(2.8)
C-1 94.9 H-2 3.72 C-2 54.5 H-2 3.84 C-2 52.0 H-2 3.78 C-2 67.9 H-3 3.94 C-3 54.7 H-3 4.08 C-3 51.3 H-3 3.94 C-3 68.8 H-4 4.01 C-4 71.9 H-4 3.48 C-4 67.5 H-4 4.08 C-4 70.8 H-5 3.23 C-5 77.8 H-5 3.97 C-5 71.8 H-5 4.42 C-5 71.1
Lipid A–P
H-1 4.35
( 8)
C-1 102.8 H-1 4.89
( 2)
C-1 92.4 H-1 5.02
( 3)
C-1 94.5 H-2 3.73 C-2 53.8 H-2 3.86 C-2 52.0 H-2 3.78 C-2 68.2 H-3 3.77 C-3 53.8 H-3 4.13 C-3 51.7 H-3 3.95 C-3 71.7 H-4 3.34 C-4 68.8 H-4 3.52 C-4 70.3 H-4 4.12 C-4 70.5 H-5 3.22 C-5 nd H-5 3.93 C-5 71.8 H-5 4.42 C-5 71.0
Fig 8 Apartial NOESY spectrum of de-O-acylated phosphorylated subfraction of lipid A The spectrum was recorded at
500 MHz and at 48 C The letters refer to the carbohydrate spin systems as was described in the text and shown in Table 2 The numerals next to the letters indicate the protons in the respective residues The inter- and intraresidue signals are labeled starting from anomeric protons Diagnostic interresidue cross peaks are underlined.
Trang 10HB-1 of the proximal DAG Both sugars possess a
anomeric configurations that are reflected in the small
values of J1,2 coupling constants and the appropriate
values of chemical shifts The downfield shift of carbon
CB-6 from the proximal DAG and strong cross peak
HC-1/HB-6a (4.39 p.p.m./3.60 p.p.m), as well as less
intensive cross peak at 4.39 p.p.m./3.89 p.p.m (HC-1/
HB-6b) on NOESY spectrum, unequivocally indicate the
presence of (1fi 6) glycosidic linkage between the two DAG residues Chemical shifts: CC-1 (103.1 p.p.m), HC-1 (4.39 p.p.m) and large ( 8Hz) coupling constants J1,2
measured for the distal DAG confirmed its b-anomeric configuration
Putting all the presented data together, we propose the chemical structures for lipid A+P(species Z, Y, X) as shown
in Fig 9
Fig 9 Tentative structures of lipid Aspecies from Mesorhizobium huakuii IFO 12543 T The proposition of the positions of 3-hydroxyl acyls is based
on preliminary chemical degradation of lipid A The predicted positions of ester bound fatty acids were elicited from literature data and specificity
of LpxXl acyltransferase [46] The proposed structures corresponds to [M + Na] + ions at m/z 2380 (Z), 2085 (Y), 1663 (X) in Fig 2B and to ions at m/z 2299 (Z1), 2004 (Y1), 1583(X1) in Fig 3B.